Illumina NovaSeq 6000 sequencing; Molecular pathology of Usher 1B patient-derived retinal organoids at single cell resolution
Molecular pathology of Usher 1B patient-derived retinal organoids at single cell resolution
Control.1 is commercially available iPSC control line purchased from WiCell. For the remaining iPSC lines, skin biopsy was collected from donor and reprogrammed to iPSC using CytoTune™-iPS 2.0 kit (Sendai virus approach). All iPSC lines subjected to differentiation using a published protocol (Gonzalez-Cordero et al., 2017 doi: 10.1016/j.stemcr.2017.07.022). retinal organoids at 35wks were collected in 1.5mL eppendorf tube and snap frozen on dry ice before stored at -80 degree until use. Briefly, when 80% confluent hPSCs were dissociated using a 1:1 dispase and collagenase solution for 10 minutes. PSC clumps were collected, washed twice with PBS and resuspended in E8 media for further maintenance culture on 6 well plates. For retinal neuroepithelial differentiation human PSCs were maintained as described above until 90-95% confluent, then media without FGF (E6, Thermo Fisher) was added to the cultures for two days (D1 and 2 of differentiation) followed by a neural induction period (up to 7 weeks) in proneural induction media (Advanced DMEM/F12, MEM non essential amino acids, N2 Supplement, 100mM Glutamine and Pen/Strep). Lightly- pigmented islands of retinal pigmented epithelium (RPE) appeared as early as week 3 in culture. Optic vesicles were formed from within the RPE region between weeks 4 and 7. During this period neuroretinal vesicles were manually excised with 21G needles and kept individually in low binding 96 well plates in retinal differentiation media (DMEM, F12, Pen/Strep and B27 without retinoic acid). The presence of immature RPE cells surrounding the optic vesicle was the criteria used to isolate NRVs. Therefore the majority of NRVs develop some pigmented RPE cells with time. RPE cells were also purified and characterised for other experiments. At 6wks of differentiation retinal differentiation medium was supplemented with 10% FBS, 100uM taurine (Sigma, T4871) and 2mM glutamax and at 10 wks 1uM retinoic acid (RA) was added. For long-term cultures, vesicles were transferred to low binding 24 well plates (5 vesicles/well) at 10 wks. At 12 wks of differentiation, in addition to B27 and other factors described above, media was supplemented with 1% N2 and the RA concentration was reduced to 0.5uM. Maintenance cultures of hPSCs were feed daily and differentiation cultures were feed every 2 days. single cell libraries were generated using the Chromium Single Cell 3' Reagent Kits v3 single cell libraries were generated using the Chromium Single Cell 3' Reagent Kits v3 according to manufacturer's instructions, Quality of libraries was tested using on Agilent TapeStation